Bridging Project 1: Conformational Transitions in P-class ATPases

P类(或E1-E2型)ATPase是一个阳离子转运酶的超家族，存在于原核生物和真核生物中，其成员调节所有常见的生物相关阳离子的膜通透性[1]。P级泵使用三磷酸腺苷逆势输送离子。肌浆网钙-ATPase(SERCA)通过交换H“将2Ca+从肌细胞胞浆泵入肌浆网。在每个正常周期中，Na/K泵以2K=的方式将3个Na“从细胞中输送到细胞内 以一个三磷酸腺苷分子的水解度为代价。这个关于P级ATPase的延伸桥梁项目代表了两个桥梁项目的结合，重点是Na/K和SERCA泵。 从SERCA的结晶学快照和最近解决的Na/K泵的第一个晶体结构，我们现在处理了一系列引人注目的图片，展示了这些酶如何看待它们运输周期的不同状态。到目前为止，SERCA是一种膜蛋白，其中最具功能的不同构象已经被精确地结构细节描述[4，5]。然而，与Na/K泵相比，使用SERCA进行电生理研究要困难得多，因为Na/K泵可以在细胞表面异源表达。因此，虽然关于Na/K泵的结构信息更有限(只有一种X射线结构可用)，但最有信息量的功能分析来自对Na/K泵的实验。 在这个提案中，我们解决了关于Na/K和SERCA泵的问题：当泵通过X射线结晶学揭示的构象状态转换时，涉及的构象动力学是什么？ ATP的结合、磷酸化和带电物种通过蛋白质核心的运动之间的耦合是什么性质？什么是阶梯式电压敏感步骤？完整的反应途径是什么？在不同的生理条件下，个体的转化率是多少？对这些问题的回答将把结构变化与膜P类ATPase泵的功能联系起来。

P-class (or E1-E2-type) ATPases constitute a superfamily of cation transport enzymes, present both in prokaryote and eukaryote, whose members mediate membrane flux of all common biologically relevant cations [1]. P-class pumps use ATP to transport ions against a gradient. The sarcoplasmic reticulum Ca+ -ATPase (SERCA) pumps 2 Ca+ from the cytosol of muscle cells to the sarcoplasmic reticulum by exchanging H"". In each normal cycle, the Na/K pump transports 3 Na" out of the cell by 2 K = into the cell at the expense of the hydrolysis of one molecule of ATP. This extended bridge project about P-class ATPases represents the combination of two bridge projects, focused on the Na/K and SERCA pumps. From crystallographic snapshots of SERCA and the recently solved first crystal structure of the Na/K pump, we now dispose of a remarkable series of pictures showing how these enzymes look at different states of their transport cycle. SERCA is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail [4, 5]. However, electrophysiological studies with SERCA are harder to do than with the Na/K pump, which can be expressed heterologously on the surface of cells. For this reason, while structural information about the Na/K pump is more limited (with only one x-ray structure available), the most informative functional analysis comes from experiments on the Na/K pump. In this proposal, we address questions about the Na/K and SERCA pumps: What are the conformational dynamics involved as the pump transits through conformational states revealed by x-ray crystallography? What is the nature of the coupling between the binding of ATP, phosphorylation, and the movements of charged species across the core of the protein? What are the stepwise voltage-sensitive steps? What is the complete reaction pathway and what are the individual transition rates under various physiological conditions? Answers to these questions will correlate the structural changes to the function of membrane P-class ATPase pumps.